key: cord-0004964-6w9n03fy authors: Sekiguchi, K.; Sugita, S.; Fukunaga, Y.; Kondo, T.; Wada, R.; Kamada, M.; Yamaguchi, S. title: Detection of equine arteritis virus (EAV) by polymerase chain reaction (PCR) and differentiation of EAV strains by restriction enzyme analysis of PCR products date: 1995 journal: Arch Virol DOI: 10.1007/bf01322675 sha: 72fc688d0a9aa57f9ca59d40f1eaa4ea89d68285 doc_id: 4964 cord_uid: 6w9n03fy A polymerase chain reaction (PCR) based assay capable of detecting and differentiating seven strains of equine arteritis virus (EAV) from around the world was developed. The primers for the PCR were chosen from the ORF 6 gene encoding the unglycosylated membrane protein (M). Viral RNA from cell culture fluids infected with each of the seven EAV strains and RNA from the live vaccine, Arvac, was detected by PCR using four sets of primers. The sensitivity of detection was increased from 100 to 1000 times by performing nested PCR enabling the detection of RNA at a level of 0.5–5 PFU. Differentiation among the virus strains and the live vaccine was achieved by cutting the PCR-amplified products from three sets of primers with six restriction endonucleases. Using this procedure it was possible to distinguish among the seven EAV strains used. A potymerase chain reaction (PCR) based assay capable of detecting and differentiating seven strains of equine arteritis virus (EAV) from around the world was developed. The primers for the PCR were chosen from the ORF 6 gene encoding the unglycosylated membrane protein (M). Viral RNA from cell culture fluids infected with each of the seven EAV strains and RNA from the live vaccine, Arvac, was detected by PCR using four sets of primers. The sensitivity of detection was increased from 100 to 1000 times by performing nested PCR enabling the detection of RNA at a level of 0.5-5 PFU. Differentiation among the virus strains and the live vaccine was achieved by cutting the PCR-amplified products from three sets of primers with six restriction endonucleases. Using this procedure it was possible to distinguish among the seven EAV strains used. The equine arteritis virus (EAV) has been classified as a member of the Togaviridae family [22, 24] . However, recently EAV has been found to resemble the coronavirus and torovirus in its genome organization and gene expression strategy [7] , yet differing from them in virion genome size and virion morphology. Therefore EAV may be a member of a recently proposed new family of viruses, the Arteriviridae, consisting of the lactate dehydrogenase-elevating virus, porcine reproductive and respiratory syndrome virus, and simian hemorrhagic fever virus [7, 21] . The clinical signs of equine viral arteritis are very variable and subclinical infections are the most common sequel [12] . To date a single serotype has been recognized, and no major antigenic variation has been demonstrated among EAV isolated from disparatechronological and geographic origins [18] . Murphy et al. showed high levels of genomic heterogeneity among isolates of EAV through oligonucleotide fingerprint comparisons [19, 20] . We determined the nucleotide sequence of O RF 6, which encodes the unglycosylated membrane protein (M), from several strains from around the world, and showed many genetic variations among the strains in spite of limited antigenic variations [23] . The objective of this study was to detect each of the EAV strains using PCR, and to differentiate the strains through restriction enzyme analysis of their PCR products, based on the M protein nucleotide sequence data of the strains, together with those of the Bucyrus [7] and modified Bucyrus strain [251. EAV strains used in this study were as follows: Bucyrus (Ohio) [8] , modified Bucyrus [9, 14] , Red Mile (Kentucky) [t 5], 84 KY-A1 (Kentucky) [19] , Wroclaw-2 (Poland) [11] , Bibuna (Switzerland) [2] and Vienna (Austria) [3] . In addition, a modified live virus vaccine (Arvac) was used. All strains of EAV were propagated in an RK 13 rabbit kidney cell line. RNA was extracted from 250 gl of culture fluid with 500 gl of RNAzol B (Biotecx Laboratories, Inc.). RNA was precipitated with isopropanol, using 2 gl of ethachinmate (Nippon gene Co., Japan) as a carrier. The RNA pellet was dissolved in 30 ILl of TE buffer (10 mM Tris-HC1, pH 8.0, 1 mM EDTA). Six primers were selected from the coding region for the M protein. The structure of the EAV genome, the location of the primer pairs and the restriction sites used are shown in Fig. 1 . Upper primer; M1, 5'-CTGAGGTATGGGAGC- These mismatches were single internal mismatches, which probably had no significant effect on the PCR product yield. The mismatches in primer M9, M14, M6 and M10 were C:A (primer: template), A:C, C:T, and G:T, respectively. These mismatches were efficiently amplified even when located at the 3' end of the primer [13] . The sensitivity of PCR for detecting EAV was determined by using serial ten-fold dilutions of infected cell fluid with a modified Bucyrus strain (2 x 107 PFU/ml). The first round PCR using the M1-M10 primer pair generated a faint though visible 449 bp product at a dilution level of 10 -3, corresponding to about 500 PFU (Fig. 3A) . When the PCR product obtained from the first round PCR was reamplified with one of the three sets of inner primers M 1-M 14, M6-M8, or M9-M 10, all of the respective 231 bp, 247 bp and 241 bp products were detected at a 10 .6 dilution level of cell fluid (Fig. 3B) . The results showed that reamplification with the nested or hemi-nested primers increased sensitivity by at least 100-1000 times, enabling us to detect at a level of 0.5-5 PFU. However, amplified DNA was not detected when the first round PCR product was amplified using an equine herpesvirus (EHV)-1 gC primer, even though a 712 bp product of EHV was produced using the primer in the presence of an EHV genome (Fig. 3B) . The genome of EHV and equine influenza virus yielded no amplification band using the EAV primer pairs. Each of the PCR products amplified using the M 1-M 14 primer was digested with each of the four restriction enzymes; Xba I, Mva I, Mbo II, and Alu I. Digestion of PCR products from the Bucyrus, modified Bucyrus, live vaccine, and Wroclaw-2 strains with Xba I gave rise to fragments of about 174 bp and 57 bp (Fig. 4a) . Products from Red Mile and 84 KY-A1 digested with Mva I produced fragments of about 131 bp and 100 bp (Fig. 4b) . Digestion of PCR products with Mbo II gave rise to fragments of about 200 bp and 31 bp in all strains except for the Wroclaw-2 and Vienna strains (Fig. 4c) . The PCR products from all strains were cleaved by digestion with Alu I into two fragments of 164 bp and 67 bp. In Bibuna strain a 164 bp fragment was further cleaved into 110 bp and 54 bp fragments, allowing for three fragments of 110 bp, 67 bp and 54 bp to be produced (Fig. 4d) . Of the PCR products using M6-M8 primer only PCR products derived from modified Bucyrus and the live vaccine were digested by Hha I, to give fragments of about t 85 bp and 62 bp (Fig. 4e) . PCR products from 84 KY-A1 and Red Mile strains were shown to share the same fragment patterns by the above 5 restriction enzymes. (Fig. 4f) . The results of the digestion of PCR products using M1-M14, M6-M8, and M9-M10, with the appropriate restriction enzymes, are summarized in Table 1 . The Bucyrus, modified Bucyrus and live vaccine, the Red Mile and 84 KY-A1, and the Wroclaw-2, Bibuna and Vienna strains were differentiated by the digestion of PCR products using the M I -M 14 primer with the four restriction enzymes XbaI, Mva I, Mbo II and AluI. The modified Bucyrus strain and live vaccine were discriminated from the others by the digestion of their PCR products using the M6-M8 primer with Hha I. Ava II digestion of PCR products using M9-M10 enabled us to distinguish between the Red Mile and 84 KY-A1 strains. In PCR based detection systems for EAV, Chirnside et al. selected primer from the leader sequence, polymerase (ORF l-b) and nucleocapsid gene (ORF 7) [4] , and Bel~tk et al. chose from the nucleocapsid gene [1] . We selected oligonucleotide primers from the O R F 6 gene because it has been shown to be an area with a higher sequence homology than O R F 7 among the members of the Arterivirus group [6, 16, 17] . We designed primer pairs so that there would be no mismatches or at most, a single base pair mismatch, and ensured that the area of amplification product contained the strain specific restriction site for the endonuclease. [5] . There were some differences in the M gene nucleotide sequence of Kentuncky 84 and Wroclaw-2 isolates from our data. If the restriction fragment length polymorphism (RFLP) patterns determined by the 6 restriction enzymes are assessed according to their nucleotide sequences, Pleurat Fluid, MAFF Ireland, 185/83, NEA V2, Kentucky 84, and Wroclaw-2 isolates should show the same RFLP patterns as the Bucyrus isolate. These isolates exhibited a 96-98% nucleotide identity with Bucyrus isolate [5] . However, our RFLP patterns were exactly as predicted from our nucleotide sequence data. In this paper we showed the RFLP patterns of single amplified PCR products from seven disparate strains using three sets of primers (Fig. 4) . The same restriction enzyme patterns were also obtained by cutting the second round PCR products with restriction enzymes (data not shown). Nested PCR and the restriction enzyme analysis of product can allow for the detection and identification of virus in small amounts of clinical samples. The live vaccine was provided after repeated passages of the modified Bucyrus strain in tissue culture. RNA from the live vaccine showed the same behavior in restriction enzyme digestion of PCR products as did the RNA from the modified Bucyrus virus. The restriction site ofHha I in the M6-M8 amplified area seems to be unique to the live vaccine, Arvac, and to the modified Bucyrus strains, according to our study and to reports by Chirnside et al. [5] . Fukunaga et al. demonstrated some serological differences between the modified Bucyrus strain and the Bucyrus, 84 KY-A1, and Wroclaw-2 strains using a serum cross neutralization test [10] . The differentiation between horses vaccinated with live vaccine and horses infected with wild strain should be reliable through RFLP analysis by using restriction sites from other ORFs, such as ORF 2, 4, and 5, in combination with serological differentiation using the neutralization. The application of PCR for EAV detection in clinical samples is presently under investigation. Evaluation of a nested PCR assay for the detection of equine arteritis virus infection The virology of equine arteritis Reverse transcription and cDNA amplification by the polymerase chain reaction of equine arteritis virus (EAV) Comparison of M and N gene sequences distinguishes variation amongst equine arteritis virus isolates Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily Isolation of a filterable agent causing arteritis of horses and abortion by mares. 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